Anti-lymphotoxin-beta receptor antibodies as anti-tumor agents

ABSTRACT

This invention relates to compositions and methods useful for treating or reducing the advancement, severity or effects of neoplasia by the administration of at least two or more lymphotoxin beta receptor (LT-β receptor) activating agents where at least one LT-β receptor activating agent is an anti-LT-β receptor antibody.

BACKGROUND OF THE INTENTION

[0001] The tumor necrosis factor (TNF) receptor family has severalmembers whose signaling can induce tumor cell death by necrosis orapoptosis (programmed cell death). The ligands TNF and lymphotoxin-α(LT-α; formerly called TNF-β) bind to and activate TNF receptors (p60and p80; herein called “TNF-R”). TNF-R signaling initiates generalimmune responses to infection or stress in normal cells, but iscytotoxic to cells with transformed phenotypes or to tumor cells. TNF-Rsignaling can selectively lyse tumor cells and virus-infected cells. Thecytotoxic effects of TNF-R signaling on tumor cells are enhanced byinterferon-γ (IFN-γ) and by a variety of conventional chemotherapeuticagents.

[0002] It would be useful to take advantage of the anti-proliferative orcytotoxic activities induced by TNF-R signaling in tumor cells fortherapeutic purposes. However, TNF-R activation has pleiotropic effectson a variety of immunoregulatory responses including the initiation ofproinflammatory cascades. Thus it has not been possible to direct thecytotoxic effects of TNF-R signaling to tumor cells withoutco-stimulating inflammatory responses which lead to general toxicity inhumans.

[0003] Similarly, stimulation of another TNF-related receptor called theFas receptor (FasR) can trigger cytotoxicity by programmed cell death ina variety of both tumor and non-tumor cell types. However, FasRactivation has been shown to cause rapid liver necrosis, thus precludingits therapeutic application in humans.

[0004] Recently, another receptor in the TNF family called the LT-βreceptor (LT-β-R) was identified (Crowe et al., Science, 264, pp. 707-10(1994)). The LT-β-R binds heteromeric lymphotoxin complexes (LT-α/β)which comprise LT-α subunits in association with another TNF-relatedpolypeptide called lymphotoxin-β (LT-β). These LT-α/β complexes aremembrane-associated and most have a LT-α1/β2 stoichiometry (Browning etal., Cell, 72, pp. 847-56 (1993); Browning et al., J. Immunol., 154, pp.33-46 (1995)).

[0005] By analogy to TNF-R and other TNF-like receptors, the activationof LT-β-R signaling is thought to occur when multiple receptors on thecell surface are brought into close proximity (Crowe et al., Science,264, pp. 707-10 (1994)). This process is referred to as receptorclustering. The TNF and LT ligands are multivalent complexes which cansimultaneously bind to and thus aggregate more than one receptor.Receptor clustering as a means for receptor activation in other systemshas been well-documented, especially for receptor tyrosine kinases(Ullrich and Schlessinger, Cell, 61, pp. 203-212 (1990); Kolanus et al.,Cell, 74, pp. 171-83 (1993)). Accordingly, administering LT-α1/β2ligands and/or LT-β-R activating agents which can induce the clusteringand downstream signaling of LT-β-R molecules on the surface of targettumor cells would be useful for directly stimulating the LT-β-R pathwayin these cells.

[0006] Signaling by LT-β-R, like TNF-R, can activate pathways that leadto cytotoxicity and cell death in tumor cells. Importantly, LT-α1/β2ligands do not bind to TNF-R with any significant affinity. For thisreason, directed LT-β-R activation in tumor cells would triggercytotoxicity in those cells without stimulating the inflammatorypathways associated with TNF-R activation. Treatment with LT-α1/β2and/or other LT-β-R activating agents would thus be useful for treatingor reducing the advancement, severity or effects of tumorigenic cells(neoplasia) while overcoming the potent side effect problems which havebeen encountered when TNF-R or FasR activation has been tried as ananti-tumor treatment.

SUMMARY OF THE INVENTION

[0007] The present invention solves the problems referred to above byproviding pharmaceutical compositions and methods for treating tumorcells by stimulating LT-β-R signaling without co-stimulatingTNF-R-associated inflammatory responses. In one embodiment, lymphotoxincomplexes formed between LT-α and multiple subunits of LT-β are provided(LT-α/β heteromeric complexes) which induce cytotoxic effects on cellsbearing the LT-β-R in the presence of a LT-β-R activating agent. Thepreferred compositions and methods of this embodiment comprise LT-α1/β2complexes in the presence of a LT-β-R activating agent. More preferably,the LT-α1/β2 complexes are in a soluble rather than a membrane-boundform, and the LT-β-R activating agent is IFN-γ.

[0008] In another embodiment of the invention, at least one antibodydirected against LT-β-R (anti-LT-β-R Ab) is used as a second LT-β-Ractivating agent in conjunction with the LT-α/β heteromeric complex. Thepreferred compositions and methods of this embodiment are characterizedby LT-α1/β2 in the presence of IFN-γ as a first activating agent, and atleast one anti-LT-β-R Ab as a second LT-β-R activating agent. Morepreferably, the LT-α1/β2 complexes are soluble and the antibody is amonoclonal antibody (anti-LT-β-R mAb).

[0009] In another embodiment of the invention, at least one anti-LT-β-RAb in the presence or absence of a second LT-β-R activating agent isused without an exogenous LT-α/β heteromeric complex. The preferredcompositions and methods of this embodiment comprise at least twoanti-LT-β-R monoclonal antibodies (anti-LT-β-R mAbs) which recognizenon-overlapping epitopes of LT-β-R in combination with IFN-γ.

[0010] In a further embodiment, this invention provides pharmaceuticalcompositions and methods for potentiating tumor cell cytotoxicitycharacterized by cross-linked anti-LT-β-R Abs used in conjunction with asecond LT-β-R activating agent. In one preferred embodiment, individualanti-LT-β-R Abs are immobilized by cross-linking them onto a surface. Inanother preferred embodiment, the anti-LT-β-R Abs are cross-linked insolution. More preferably, the anti-LT-β-R Abs are monoclonal antibodiesand the second LT-β-R activating agent is IFN-γ.

[0011] This invention further provides a novel screening process forselecting LT-β-R activating agents, such as anti-LT-β-R Abs, thatfunction in combination with LT-α/β heteromeric complexes to promotetumor cell death. The assay makes use of the increased sensitivity ofhuman adenocarcinoma cells to LT-α/β heteromeric complexes in thepresence of LT-β-R activating agents in a cytotoxicity assay. Theprocedure used to test putative LT-β-R activating agents is exemplifiedfor the case of anti-LT-β-R antibodies, and comprises the followingsteps:

[0012] 1) Tumor cells (e.g., HT29 human adenocarcinoma cells) arecultured for several days in media containing IFN-γ and purifiedLT-α1/β2 in the presence or absence of the particular anti-LT-β-R Abbeing assayed;

[0013] 2) The cells are treated with a dye that stains living cells; and

[0014] 3) The number of stained cells is quantitated to determine thefraction of tumor cells killed in the presence of the LT-α1/β2, IFN-γand the test anti-LT-β-R Ab in each sample. Alternatively, the number ofsurviving cells can be determined by any of a number of well-knownassays which measure cell viability, such as ³H-thymidine incorporationinto DNA.

[0015] An anti-LT-β-R Ab (or an Ab combination) which significantlyincreases the percentage of tumor cells killed in this assay is a LT-β-Ractivating agent within the scope of this invention. This cytolyticassay can be adapted to identify new LT-β-R activating agents whichfunction in combination with LT-α/β heteromeric complexes.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1A. Sizing analyses of the purified LT-α/β heteromericcomplex forms separated by TNF-R and LT-β-R immunoaffinitychromatography. Purified proteins were analyzed on a TSK 3000 HPLC resinin phosphate-buffered saline. The position of various size markers isshown.

[0017]FIG. 1B. Ion exchange analyses of purified LT forms using a Poroscarboxymethyl column (4.6 mm×100 mm) on a BioCad instrument (PerceptiveBiosystems). 27 μg of each sample protein was loaded onto a column andeluted in a gradient of 0 to 1M NaCl over 20 column volumes at 5 ml/minin a Buffer containing 16.66 μM Hepes, 16.66 μM Na Acetate, and 16.66 μMMes buffer (pH 6.5).

[0018]FIG. 2A. Comparison of the cytotoxic activity of: Anti-Fasreceptor mAb CH-11 (--); TNF (-∘-); LT-α (-□-); LT-α1/β2 (-▪-); andLT-α2/β1 (-♦-) on human adenocarcinoma HT29 cells in either the presenceor absence of 80 U/ml IFN-γ.

[0019]FIG. 2B. Comparison of the ability of 5 μg/ml of human IgG (--),soluble p60TNF-R-Fc (-∘-) and soluble LT-β-R-Fc receptor-immunoglobulinchimeras (-□-) to inhibit the cytotoxic effects in FIG. 2A in thepresence of 80 U/ml IFN-γ.

[0020]FIG. 3. Anti-LT-β-R mAbs potentiate the cytotoxic effect of LT-α1/β2 on human adenocarcinoma HT29 cells. (A) The LT-α1 /β2 cytolyticeffects on HT29 cells are potentiated by the presence of anti-LT-β-R mAbCDH10. LT-α1/β2 effects were measured without mAb (--), and in thepresence of 0.5 μg/ml control IgG1 (-▪-), 0.05 μg/ml CDH10 (-∘-) and 0.5μg/ml (-□-) CDH10. (B) The LT-α1/β2 cytolytic effects on HT29 cells areinhibited by the presence of the anti-LT-β-R mAb BDA8. LT-α1/β2 effectswere measured in the presence of 2 μg/ml control IgG1 (-▪-) oranti-LT-β-R mAb BDA8 (-□-). The difference between the behavior of theCDH10 and BDA8 anti-LT-β-R mAbs in this assay is one indication thatthey are directed to different epitopes of the LT-β-R.

[0021]FIG. 4. Immobilized anti-LT-β-R mAbs are cytotoxic to humanadenocarcinoma HT29 cells. (A) The anti-LT-β-R mAbs have a directcytotoxic effect on HT29 cells when they are immobilized on a surface.Plates were coated with IgG1 (--), a mAb directed against an unrelatedabundant cell surface antigen HT29/26 (-▪-), BDA8 (-∘-) and CDH10 (-□-).(B) The effects of soluble anti-LT-β-R mAbs alone on the growth of HT29cells. Symbols as in (A). These anti-LT-β-R mAbs in their soluble formdo not have significant cytotoxic effects on HT29 cells whenadministered individually.

[0022]FIG. 5. Representative quantitation of the enhanced cytotoxicityto tumor cells by treating with pairs of soluble anti-LT-β-R mAbs. (A)The cytotoxic effects on HT29 cells of control IgG1 (100 ng/ml),anti-LT-β-R mAb BHA10 (100 ng/ml), anti-LT-β-R mAb CBE11 (50 ng/ml),BHA10 (100 ng/ml)+IgG (100 ng/ml), and BHA10 (100 ng/ml)+CBE11 (50ng/ml). IFN-γ was present at 80 U/ml. (B) The cytotoxic effects on HT29cells of control IgG1 (100 ng/ml), anti-LT-β-R mAb CDH10 (100 ng/ml),anti-LT-β-R mAb CBE11 (50 ng/ml), CDH10 (100 ng/ml)+IgG1 (100 ng/ml),and CDH10 (100 ng/ml)+CBE11 (50 ng/ml). IFN-γ was present at 80 U/ml.(C) The cytotoxic effects on HT29 cells of control IgG1 (100 ng/ml),anti-LT-β-R mAb CDH10 (33 ng/ml), anti-LT-β-R mAb AGH1 (50 ng/ml), andCDH10 (33 ng/ml)+AGH1 (50 ng/ml) on HT29 cells IFN-γ was present at 80U/ml. (D) As in (C), except that WiDr human adenocarcinoma cells wereused in the cytolytic assay (Raitano and Korc, J. Biol. Chem., 265, pp.10466-472 (1990)).

[0023]FIG. 6. Tumor size in SCID mice treated with an anti-LT-β-R mAb.(A) Size of the human adenocarcinoma WiDr tumor in SCID mice 30 daysafter inoculation with an antibody co-treatment. Mice were treated ondays 1 and 2 with saline, IFN-γ alone, an anti-LT-β-R mAb (CBE11) withand without IFN-γ and a control anti-human LFA-3 mAb (1E6) with IFN-γ.The mean of each group is indicated by a crossbar. Means, standarddeviations, and number of animals (in parentheticals) for the fivegroups (left to right) were: 0.88+/−0.59 (14), 1.21+/−0.7 (21),0.041+/−0.052 (16), 0.11+/−0.1 (12), and 0.98+/−1.16 (12). (B) Size ofthe human adenocarcinoma WiDr tumor in SCID mice from 14 to 49 daysafter tumor cell inoculation with a 15 day post-inoculation antibodytreatment. Tumors were grown to an average diameter of 0.53 cm (0.076cc) without any treatment and i.p. injections were started on day 15 andcontinued as indicated by the arrows. Means and standard deviations areindicated for a group of 12 animals treated either with IFN-γ alone(1×10⁶ U/injection) (-□-), IFN-γ with 50 μg 1E6 anti-LFA-3 mAb (-∘-),IFN-γ with 50 μg CBE11 anti-LT-β-R mAb (-Δ-) or 50 μg CBE11 anti-LT-β-RmAb alone (not shown).

DETAILED DESCRIPTION OF THE INVENTION

[0024] In order that the invention herein described may be fullyunderstood, the following detailed description is set forth.

[0025] The term “anti-tumor activity” refers to the ability of asubstance or composition to block the proliferation of, or to induce thedeath of tumor cells which interact with that substance or composition.

[0026] The term “apoptosis” refers to a process of programmed celldeath.

[0027] The term “cytotoxic activity” refers to the ability of asubstance or composition to induce the death of cells which interactwith that substance or composition.

[0028] The term “epitope” (or antigenic determinant) is defined as thepart of a molecule that combines with a single antigen binding site onan antibody molecule. A single epitope is recognized by a monoclonalantibody (mAb). Multiple epitopes are normally recognized by polyclonalantibodies (Ab).

[0029] The “Fc domain” of an antibody refers to a part of the moleculecomprising the CH2, CH3 and hinge regions but lacking the antigenbinding sites.

[0030] The term “interferon inducing agent” refers to any agent which iscapable of directly or indirectly stimulating the endogenous productionof either type I (IFN-α, IFN-β) or type II (IFN-γ) interferons. Examplesof interferon inducing agents include double stranded RNA molecules, anda variety of plant or pharmaceutically-derived compounds.

[0031] The terms “LT-α mutein” and “LT-β mutein” refer to LT-α or LT-βpolypeptides having one or more amino acid changes compared to the aminoacid sequence of the corresponding native polypeptide.

[0032] The term “LT-β-R activating agent” refers to any agent which canaugment ligand binding to LT-β-R, cell surface LT-β-R clustering orLT-β-R signaling, or which can influence how the LT-β-R signal isinterpreted within the cell. Examples of LT-β-R activating agentsinclude IFN-α, IFN-γ, TNF, interferon inducing agents, solubleanti-LT-β-R Abs, cross-linked anti-LT-β-R Abs and multivalentanti-LT-β-R Abs.

[0033] The term “LT-β-R signaling” refers to all molecular reactionsassociated with the LT-β-R pathway and subsequent molecular reactionswhich result therefrom.

[0034] The term “anti-LT-β-receptor antibody” (“anti-LT-β-R Ab”) refersto any antibody that recognizes and binds to at least one epitope of theLT-β receptor.

[0035] The term “anti-LT-β receptor monoclonal antibody” (“anti-LT-β-RmAb”) refers to any monoclonal antibody that recognizes and binds to asingle epitope of the LT-β-R.

[0036] The term “cross-linked anti-LT-β-R (m)Abs” refer to antibodiesdirected against the LT-β-R which have either been cross-linked to eachother to form antibody agglomerates in solution using an anti-LT-β-Rantibody (Ab) or (mAb) cross-linking agent, or which have beenimmobilized in close proximity to one another on a surface or matrix.

[0037] The term “anti-LT-β-R Ab (or mAb) cross-linking agent” refers toany agent which can covalently or non-covalently aggregate anti-LT-β-RAbs in solution so that the Abs can bind to and potentiate target cellsurface LT-β receptor clustering. Such cross-linking agents include butare not limited to chemical cross-linking agents, secondary antibodieswhich react with portions of the anti-LT-β-R Abs or mAbs, and soluble orsurface-bound Fc receptors—either endogenous or added exogenously—whichcan bind to anti-LT-β-R Abs.

[0038] The terms “LT-α biological activity”, “LT-β biological activity”,and “LT-α/β biological activity” are defined as: 1) immunologicalcross-reactivity with an antibody directed against at least one epitopeof the corresponding native subunit or complex of subunits; or 2) theability of the LT subunit or complex of subunits to compete for ligandbinding sites on a LT-specific receptor such as TNF-R or LT-β-R; or 3)having the ability to stimulate an immune regulatory response orcytotoxic activity qualitatively in common with a native LT subunit orcomplex.

[0039] The term “LT-α/β heteromeric complex” refers to a stableassociation between at least one LT-α subunit and more than one LT-βsubunits. The subunits can associate through electrostatic, van derWaals, or covalent interactions. Preferably, the LT-α/β heteromericcomplex has at least two adjacent LT-β subunits and lacks adjacent LT-αsubunits. Most preferably, the complex has the stoichiometry LT-α1/β2.

[0040] The term “multivalent ligand” refers to a molecule or complexwhich has more than one receptor binding site and which is capable ofsimultaneously binding and bringing into close proximity at least tworeceptor molecules.

[0041] A “type I leader sequence” is an amino-terminal portion of aeukaryotic protein which serves as a signal to direct the protein to theendoplasmic reticular (ER) membrane and often through the entiresecretion pathway. The leader sequence is usually cleaved off by asignal peptidase in the ER membrane.

[0042] A “signal sequence” is the functional equivalent of a eukaryotictype I leader sequence in prokaryotic hosts, and directs thetranslocation of proteins into or across lipid bilayer membranes of abacterium.

[0043] A “soluble LT-α/β heteromeric complex” is a LT-α/β heteromericcomplex comprising soluble LT-β subunits, wherein the amino acidsequences which localize the polypeptide to the membrane have beendeleted or inactivated, rendering the LT-β subunit soluble. SolubleLT-α/β heteromeric complexes can be secreted by an appropriate host cellwhich has been engineered to express both subunits.

[0044] A “surface LT-α/β complex” is a complex comprising LT-α andmembrane-bound LT-β subunits which is displayed on the cell surface.

[0045] Production of Membrane-bound LT-α/β Complexes

[0046] Cell surface lymphotoxin complexes have been characterized inCD4⁺ T cell hybridoma cells (II-23.D7) that express high levels of LT(Browning et al., J. Immunol., 147, pp. 1230-37 (1991); Androlewicz etal., J. Biol. Chem., 267, pp. 2542-47 (1992)). Mature LT-α lacks atransmembrane domain and is localized to the cell surface throughinteraction with at least one membrane-bound LT-β subunit.Membrane-bound (surface) LT-α/β heteromeric complexes have predominantlya LT-α1/β2 stoichiometry.

[0047] LT-β as a cell membrane protein binds LT-α during synthesis, thus“targeting” the LT-α to the cell membrane. In the absence of LT-β, LT-αis secreted into the extracellular medium. LT subunits normally assembleinto complexes inside the cell prior to protein export into themembrane. Once LT-β subunits are inserted into the membrane, they do notform stable complexes with secreted LT-α. Thus if the membrane-boundform of a LT-α/β heteromeric complex is desired, it is preferable toco-express the desired LT-α and LT-β subunits within the same cell.

[0048] The surface LT-α/β heteromeric complex can be reconstructed byco-transfection of host cells with both the LT-α and LT-β genes. SurfaceLT complexes cannot be observed on stable cell lines which expresseither LT gene alone. However, if the host cell normally produces largeamounts of LT-α (e.g. RPMI 1788 cells; see below), then transfectionwith a LT-β gene which encodes the desired LT-β polypeptide should besufficient to generate LT-α/β complexes comprising full-length LT-αsubunits.

[0049] Co-expression of LT-α and LT-β polypeptides in a number ofeukaryotic expression systems leads to their assembly and export asactive ligand (Crowe et al., J. Immunol. Methods, 168, 79-89 (1994)).Host systems that can be used include but are not limited to CHO cells,COS cells, B cells including myelomas, baculovirus-infected insect cellsand yeast.

[0050] The LT-α subunit of the LT-α/β heteromeric complexes of thisinvention can be selected from lymphotoxin-α, native human or animallymphotoxin-α, recombinant lymphotoxin-α, soluble lymphotoxin-α,secreted lymphotoxin-α, lymphotoxin-α muteins having LT-α biologicalactivity, or lymphotoxin-α fragments of any of the above having LT-αbiological activity.

[0051] The LT-α polypeptide can be any soluble form of the moleculeincluding active fragments thereof which can be produced in eukaryoticexpression systems, wherein the natural LT-α leader sequence will becleaved off. Alternatively, fusions of the mature LT-α sequence with aheterologous signal sequence can be used to maximize the secretion ofLT-α in other host systems. Signals are chosen based on the intendedhost cell, and may include bacterial, yeast, mammalian and viralsequences. The native signal, or the vascular cell adhesion molecule-1(VCAM-1) signal sequence is suitable for use in mammalian expressionsystems.

[0052] LT-α polypeptides can also be fused to polypeptides having aprolonged plasma half-life such as immunoglobulin chains or fragmentsthereof. Plasma proteins which may be used to enhance plasma half-lifeinclude serum albumin, immunoglobulins, apolipoproteins, andtransferrin. Polyethylene glycol (PEG) attachment may stabilize thepolypeptide and lower its immunogenicity. Preferably the LT-α fusionprotein is not significantly immunogenic in the subject to be treatedand the plasma protein does not cause undesirable side effects insubjects due to its normal biological activity.

[0053] Human LT-α is glycosylated on N and O residues, and depending onthe source, exhibits considerable sugar-based microheterogeneity. Theoligosaccharide composition of the particular LT-α chosen to form the LTcomplex may affect in vivo clearance rates (Fukushima et al., Arch.Biochem. Biophys., 304, pp. 144-53 (1993)). Since glycosylation variantscan be produced by expression in different host cells, this is onefactor to be considered in selecting a source of LT-α.

[0054] LT-α can be purified from a B lymphoblastoid line RPMI 1788,which constitutively secretes LT-α and which can be induced to secretehigher levels by treating with the phorbol ester PMA (Aggarwal et al.,J. Biol. Chem., 259, pp. 686-91 (1984)). Alternatively, the cloned humanLT-α gene can be used to recombinantly produce LT-α polypeptides indifferent host systems including bacteria (Schoenfeld et al., J. Biol.Chem., 266, pp. 3863-69 (1991)); baculovirus-infected insect cells(Crowe et al., J. Immunol. Methods, 168, pp. 70-89 (1994)); andmammalian cells (Browning and Ribolini, J. Immunol., 143, pp. 1859-67(1989); Fukushima et al., Arch. Biochem. Biophys., 304, pp. 144-53(1993)).

[0055] Portions of the LT-α gene which encode polypeptide fragmentshaving LT-α biological activity can be evaluated using routine screeningassays. Useful screening assays for LT-α biological activity includecompetitive inhibition assays with native LT-α bound to TNF-R, ormeasuring either directly or indirectly by inhibition the ability of theLT-α to induce cytotoxicity of tumor cells in assays known to the art.Preferably, LT-α fragments are assembled into heteromeric complexes withLT-β and the complexes assayed for LT-α/β biological activity bycompetitive inhibition with LT-α/β bound to LT-β-R, or for their abilityto induce cytotoxicity of tumor cells in the assays disclosed herein.

[0056] Lymphotoxin-β, also referred to as p33, has been identified onthe surface of T lymphocytes, T cell lines, B cell lines andlymphokine-activated killer cells. LT-β is the subject of applicants'co-pending international applications PCT/US91/04588, published Jan. 9,1992 as WO 92/00329; and PCT/US93/11669, published Jun. 23, 1994 as WO94/13808, which are herein incorporated by reference.

[0057] The LT-β gene encodes a polypeptide of 240-244 amino acids(Browning et al., Cell, 72, pp. 847-56 (1993)). LT-β is a type IImembrane protein with a short N-terminal cytoplasmic domain followed bya membrane anchoring domain of 30 hydrophobic amino acids. It has asingle N-linked glycosylation site and has only one cysteine residuewhich does not appear to be involved in intersubunit disulfide bondformation.

[0058] The LT-β subunits comprising the LT-α/β heteromeric complexes ofthe present invention can be selected from lymphotoxin-β, native humanor animal lymphotoxin-β, recombinant lymphotoxin-β, solublelymphotoxin-β, secreted lymphotoxin-β, lymphotoxin-β muteins having LT-βbiological activity, or lymphotoxin-β fragments of any of the abovehaving LT-β biological activity.

[0059] As discussed above for the LT-α polypeptide, the LT-βpolypeptides can also be modified to increase their solubility or plasmahalf-life using the same methods. Likewise, portions of the LT-β genewhich encode polypeptide fragments having LT-β biological activity canbe evaluated using routine screening assays as discussed for LT-α.

[0060] Production of Soluble Complexes

[0061] Soluble (non-membrane-bound) LT-α/β heteromeric complexescomprise LT-β subunits which have been changed from a membrane-bound toa soluble form. These complexes are described in detail in applicants'co-pending international application (PCT/US93/11669, published Jan. 9,1992 as WO 94/13808). Soluble LT-β peptides are defined by the aminoacid sequence of lymphotoxin-β wherein the sequence is cleaved at anypoint between the end of the transmembrane region (i.e. at about aminoacid #44) and the first TNF homology region (i.e. at amino acid #88)according to the numbering system of Browning et al., Cell, 72, pp.847-56 (1993).

[0062] Soluble LT-β polypeptides may be produced by truncating theN-terminus of LT-β to remove the cytoplasmic tail and transmembraneregion (Crowe et al., Science, 264, pp. 707-710 (1994)). Alternatively,the transmembrane domain may be inactivated by deletion, or bysubstitution of the normally hydrophobic amino acid residues whichcomprise a transmembrane domain with hydrophilic ones. In either case, asubstantially hydrophilic hydropathy profile is created which willreduce lipid affinity and improve aqueous solubility. Deletion of thetransmembrane domain is preferred over substitution with hydrophilicamino acid residues because it avoids introducing potentiallyimmunogenic epitopes.

[0063] The deleted or inactivated transmembrane domain may be replacedwith or attached to a type I leader sequence (e.g. the VCAM-1 leader)such that the protein is secreted beginning with a sequence anywherefrom between val40 to pro88. Soluble LT-β polypeptides may include anynumber of well-known leader sequences at the N-terminus. Such a sequencewould allow the peptides to be expressed and targeted to the secretionpathway in a eukaryotic system. See, e.g., Ernst et al., U.S. Pat. No.5,082,783 (1992).

[0064] Soluble LT-α/β heteromeric complexes may be produced byco-transfecting a suitable host cell with DNA encoding LT-α and solubleLT-β (Crowe et al., J. Immunol. Methods, 168, pp. 79-89 (1994)). SolubleLT-β secreted in the absence of LT-α is highly oligomerized. However,when co-expressed with LT-α, a 70 kDa trimeric-like structure is formedwhich contains both proteins. It is also possible to produce solubleLT-α1/β2 heteromeric complexes by transfecting a cell line whichnormally expresses only LT-α (such as the RPMI 1788 cells discussedabove) with a gene encoding a soluble LT-β polypeptide.

[0065] LT-α and LT-β polypeptides may be separately synthesized,denatured using mild detergents, mixed together and renatured byremoving the detergent to form mixed LT heteromeric complexes which canbe separated (see below).

[0066] Purification of LT-α1/β2 Complexes

[0067] Soluble LT-α1/β2 heteromeric complexes are separated fromco-expression complexes comprising a different subunit stoichiometry bychromatography using TNF and LT-β receptors as affinity purificationreagents. The TNF receptors only bind within α/α clefts of LT complexes.The LT-β receptor binds with high affinity to β/β clefts, and with loweraffinity to α/β clefts of heteromeric LT-α/β complexes. Accordingly,LT-α3 and LT-α2/β1 will bind to TNF-R. The LT-β-R can also bind LT-α2/β1trimers (within the α/β clefts) but cannot bind LT-α3. In addition, theLT-β-R (but not TNF-R) binds LT-α1/β2 and LT-βn (the exact compositionof such preparation is unknown, however, they are large aggregates).

[0068] The receptor affinity reagents can be prepared as either asoluble extracellular domain (see for example Loetscher et al., J. Biol.Chem., 266, pp. 18324-29 (1991)), or as chimeric proteins with theextracellular ligand binding domain coupled to an immunoglobulin Fcdomain (Loetscher et al., J. Biol Chem., 266, pp. 18324-29 (1991); Croweet al., Science, 264, pp. 707-710 (1994)). Receptors are coupled toaffinity matrices by chemical cross-linking using routine procedures.

[0069] There are two schemes by which the LT-α1/β2 ligand can bepurified using receptors and immunoaffinity chromatography. In the firstscheme, a supernatant from an appropriate expression systemco-expressing both LT-α and the truncated LT-β form is passed over aTNF-R column. The TNF-R will bind LT-α3 and LT-α2/β1 trimers. The flowthrough from the TNF-R column will contain LT-β (n) and LT-α1/β2.

[0070] In the second scheme, all LT-β -containing forms (LT-β (n),LT-α1/β2 and LT-α2/β1) are bound to and eluted from a LT-β-R columnusing classical methods such as chaotrophe or pH change. (LT-α3 flowsthrough this column). The eluate is neutralized or the chaotropheremoved, and the eluate is then passed over a TNF-R column, which bindsonly to the LT-α2/β1 trimers. The flow through of this column willcontain LT-β (n) and LT-α1/β2 trimers.

[0071] In both cases, pure LT-α1/β2 trimers can be separated from LT-βby subsequent gel filtration and/or ion exchange chromatographicprocedures known to the art.

[0072] Alternatively, different forms of LT-α/β heteromeric complexescan be separated and purified by a variety of conventionalchromatographic means. It may also be preferable to combine a series ofconventional purification schemes with one of the immunoaffinitypurification steps described above.

[0073] Source of Anti-LT-β-R Antibodies

[0074] Polyclonal antibody sera directed against the human LT-β receptorare prepared using conventional techniques by injecting animals such asgoats, rabbits or mice subcutaneously with a human LT-β receptor-Fcfusion protein (Example 2) in complete Freund's adjuvant, followed bybooster intraperitoneal or subcutaneous injection in complete Freunds.Polyclonal antisera containing the desired antibodies which are directedagainst the LT-β receptor are screened by conventional procedures.

[0075] Mouse monoclonal antibodies (mAbs) directed against a human LT-βreceptor-Fc fusion protein are prepared by intraperitoneal immunizationof RBF mice repetitively with a CHO cell-derived recombinant LT-βreceptor-Fc fusion protein (LT-β-R-Fc) attached to protein A sepharosebeads in the absence of adjuvant. Animals are finally boosted withsoluble LT-β-R-Fc (both i.p. and i.v.), spleen cells are fused usingclassical protocols, and hybridomas are screened by ELISA (Ling et al.,J. Interferon and Cytokine Res., 15, pp. 53-59 (1995)). Hybridomas arefurther screened for their ability to block binding of activated II-23hybridoma cells—which express surface LT-α1/β2—to LT-β-R-Fc-coatedplates in a cell panning assay. Pure mAbs are prepared by protein Asepharose purification of IgG from hybridoma culture supernatants.

[0076] Various forms of anti-LT-β-R antibodies can also be made usingstandard recombinant DNA techniques (Winter and Milstein, Nature, 349,pp. 293-99 (1991)). For example, “chimeric” antibodies can beconstructed in which the antigen binding domain from an animal antibodyis linked to a human constant domain (e.g. Cabilly et al., U.S. Pat. No.4,816,567; Morrison et al.,Proc. Natl. Acad. Sci. U.S.A., 81, pp.6851-55 (1984)). Chimeric antibodies reduce the observed immunogenicresponses elicited by animal antibodies when used in human clinicaltreatments.

[0077] In addition, recombinant “humanized antibodies” which recognizethe LT-β-R can be synthesized. Humanized antibodies are chimerascomprising mostly human IgG sequences into which the regions responsiblefor specific antigen-binding have been inserted (e.g. WO 94/04679).Animals are immunized with the desired antigen, the correspondingantibodies are isolated, and the portion of the variable regionsequences responsible for specific antigen binding are removed. Theanimal-derived antigen binding regions are then cloned into theappropriate position of human antibody genes in which the antigenbinding regions have been deleted. Humanized antibodies minimize the useof heterologous (inter-species) sequences in human antibodies, and areless likely to elicit immune responses in the treated subject.

[0078] Construction of different classes of recombinant anti-LT-β-Rantibodies can also be accomplished by making chimeric or humanizedantibodies comprising the anti-LT-β-R variable domains and humanconstant domains (CH1, CH2, CH3) isolated from different classes ofimmunoglobulins. For example, anti-LT-β-R IgM antibodies with increasedantigen binding site valencies can be recombinantly produced by cloningthe antigen binding site into vectors carrying the human μ chainconstant regions (Arulanandam et al., J. Exp. Med., 177, pp. 1439-50(1993); Lane et al., Eur. J. Immunol., 22, pp. 2573-78 (1993);Traunecker et al., Nature, 339, pp. 68-70 (1989)).

[0079] In addition, standard recombinant DNA techniques can be used toalter the binding affinities of recombinant antibodies with theirantigens by altering amino acid residues in the vicinity of the antigenbinding sites. The antigen binding affinity of a humanized antibody canbe increased by mutagenesis based on molecular modeling (Queen et al.,Proc. Natl. Acad. Sci. U.S.A., 86, pp. 10029-33 (1989); WO 94/04679).

[0080] It may be desirable to increase or to decrease the affinity ofanti-LT-β-R Abs for the LT-β-R depending on the targeted tissue type orthe particular treatment schedule envisioned. For example, it may beadvantageous to treat a patient with constant levels of anti-LT-β-R Abswith reduced ability to signal through the LT-β pathway forsemi-prophylactic treatments. Likewise, anti-LT-β-R Abs with increasedaffinity for the LT-β-R may be advantageous for short-term,tumor-targeted treatments.

[0081] Screening Anti-LT-β-R Antibodies for LT-β-R Activating Agents

[0082] The anti-LT-β-R antibodies of this invention can potentiate theanti-tumor activity of LT-α/β heteromeric complexes (preferablyLT-α1/β2) in the presence of an LT-β-R activating agent such as IFN-γ.These anti-LT-β-R antibodies are also referred to herein as LT-β-Ractivating agents. The antibodies which act as LT-β-R activating agentsare selected as follows:

[0083] 1) A series of tissue culture wells containing tumor cells suchas HT29 cells are cultured for three to four days in media containing aLT-β-R activating agent such as IFN-γ, and purified LT-α/β heteromericcomplex—preferably LT-α1/β2—in the presence or absence of serialdilutions of the anti-LT-β-R Ab being tested;

[0084] 2) A vital dye stain which measures mitochondrial function suchas MTT is added to the cell mixture and reacted for several hours;

[0085] 3) The optical density of the mixture in each well is quantitatedat 550 nm wavelength light (OD 550). The OD 550 is inverselyproportional to the number of tumor cells killed in the presence of theLT-α/β heteromeric complex, the LT-β-R activating agent and testanti-LT-β-R Ab in each well.

[0086] The preferred antibodies of this invention which act individuallyas LT-β-R activating agents in the presence of LT-α1/β2 and IFN-γinclude the BKA11, CDH10, BHA10 and BCG6 anti-LT-β-R mAbs (Table 2,infra).

[0087] Cross-linking Anti-LT-β-R Antibodies

[0088] The cross-linked anti-LT-β-R antibodies of this invention actindividually as LT-β-R activating agents without exogenous LT-α/βheteromeric complexes in the presence of a second LT-β-R activatingagent such as IFN-γ. Cross-linked anti-LT-β-R Abs apparently bind to andinduce clustering of cell surface LT-β receptors thereby activating LT-βreceptor-mediated targeted cell death.

[0089] In one embodiment, one or more types of anti-LT-β-R antibodiesare cross-linked by immobilization onto a water-insoluble matrix orsurface. Derivatization with a bifunctional agent is useful forcross-linking the antibodies to the water-insoluble support matrix orsurface. Agents commonly used to effect cross-linking of antibodies to awater insoluble support matrix or surface include 1,1bis(-diazoacetyl)-2-phenylethane, glutyraldehyde, N-hydroxysuccinamideesters including esters with 4-azidosalicylic acid, homobifunctionalimidoesters including disuccinimidyl esters, and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane. Derivatizing agents such asmethyl-3-[(p-azidophenyl) dithio] propioimidate form photoactivatableintermediates which can be selectively cross-linked when stimulated withlight. Reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the substrates described in U.S.Pat. Nos. 3,959,080; 3,969,287; 3,691,016; 4,195,128; 4,247,642;4,229,537; 4,055,635; and 4,330,440 can also be used for proteinimmobilization and cross-linking.

[0090] The surfaces to which the antibodies are attached can benon-proteinaceous polymer, usually a hydrophilic polymer either fromnatural or synthetic sources. Hydrophilic polyvinyl polymers such aspolyvinylalcohol (PVA) and polyvinylpyrrolidone (PVP) can be used. Alsouseful are polyalkylene ethers such as polyethylene glycol,polypropylene glycol, polyoxyethylene esters or methoxy polyethyleneglycol, polyoxyalkylenes such as polyoxyethylene and polyoxypropylene,and block copolymers of polyoxyethylene and polyoxypropylene(Pluronics); polymethacrylates; carbomers; branched or unbranchedpolysaccharides which comprise the saccharide monomers D-mannose, D- andL-galactose, fucose, fructose, D-xylose, L-arabinose, D-glucuronic acid,sialic acid, D-galacturonic acid, D-mannuronic acid (e.g.poly-mannuronic acid or alginic acid), D-glucosamine, D-galactosamine,D-glucose and neuraminic acid including homopolysaccharides andheteropolysaccharides such as lactose, amylopectin, starch, hydroxyethylstarch, amylose, dextran sulfate, dextran, dextrins, glycogen, or thepolysaccharide subunit of acid mucopoly-saccharides, e.g. hyaluronicacid; polymers of sugar alcohols such as polysorbitol and polymannitol;and heparin or heparon.

[0091] The polymer prior to cross-linking is preferably water solubleand preferably contains only a singly reactive chemical group to avoidmultiple cross-linking events with the antibody. In any case, reactionconditions should be optimized to reduce cross-linking and to recoverproducts—either directly or by a subsequent gel filtration orchromatographic step—having a substantially homogenous molecular weightrange. The optimal molecular weight of the cross-linked antibody matrixwill be determined by routine experimentation using the cytotoxicity andreceptor binding assays disclosed herein.

[0092] The final conjugate after cross-linking is preferably soluble inphysiological fluids such as blood. The polymer should not be highlyimmunogenic in the conjugate form, and should possess a viscositycompatible with intravenous infusion or injection if either is anintended route of administration.

[0093] The polymer may also be water insoluble. Materials which may beused include hydrophilic gels, or shaped articles having surfaces towhich the antibodies can be immobilized such as surgical tubing,catheters, or drainage conduits. It is preferable to use solid supportmaterials which are biologically compatible and substantially inert inphysiological surroundings. A material is biologically compatible if itdoes not substantially stimulate immune responses includinginflammation, or attract fibrotic cells when placed inside the body of asubject.

[0094] Anti-LT-β-R Abs may also be immobilized onto surfaces which havebeen covalently or non-covalently coated with secondary antibodies thatwill bind to the primary anti-LT-β-R Abs (e.g., goat anti-mouse IgGantibodies; see Example 7). Each anti-LT-β-R mAb tested individually,when immobilized onto a surface with secondary antibodies, acts as anLT-β-R activating agent in the presence of IFN-γ (FIGS. 4 and 7).

[0095] In an alternative embodiment, cross-linked anti-LT-β-R Abs insolution act as LT-β-R activating agents. Anti-LT-β-R Abs can becross-linked by means of an anti-LT-β-R Ab (or mAb) cross-linking agent.An anti-LT-β-R Ab (or mAb) cross-linking agent according to thisinvention is any agent capable of either covalently linking, or ofnon-covalently aggregating the anti-LT-β-R Abs (or mAbs) in solution sothat the cross-linked anti-LT-β-R Abs (or mAbs) can bind to andpotentiate target cell surface LT-β-R clustering. Such anti-LT-β-R Ab(or mAb) cross-linking agents include but are not limited to chemicalcross-linking reagents which can be reacted with the antibodies in acontrolled manner as described above. Alternatively, secondaryantibodies, Sepharose A, Fc receptors, or other agents that will bind toand aggregate multiple primary anti-LT-β-R Abs without blocking theiractivity can be used to form anti-LT-β-R Abs agglomerates in solution.

[0096] Multiple Anti-LT-β-R Abs in Solution Act as LT-β-R ActivatingAgents

[0097] Compositions comprising multiple anti-LT-β-R Abs in solutionwhich act as LT-β-R activating agents by potentiating surface LT-β-Rclustering are provided by this invention. Polyclonal anti-LT-β-R Absdirected against different epitopes of the LT-β-R can be used.Preferably, the anti-LT-β-R Abs are monoclonal Abs directed againstdifferent and non-overlapping epitopes of the LT-β-R.

[0098] The combined anti-LT-β-R mAb approach to LT-β receptor activationrequires combining two non-overlapping epitopes. Moreover, it is likelythat productive receptor aggregation is only achieved with certainepitopes. We have identified the presence of at least four unique LT-β-Rimmunoreactive epitopes. Additional epitopes (as defined by new mAbs)may be identified by continuing to fuse immunized mouse spleen cells, byimmunizing different species of animals, and by using different routesof immunization.

[0099] Epitopes can also be directly mapped by assessing the ability ofdifferent mAbs to compete with each other for binding to the LT-β-Rusing BIAcore chromatographic techniques (Pharmacia BIAtechnologyHandbook, “Epitope Mapping”, Section 6.3.2, (May 1994); see also Johneet al., J. Immunol. Methods, 160, pp. 191-8 (1993)).

[0100] Individual LT-β-R mAbs can be grouped into at least four classesaccording to their ability to cooperate in combination with other LT-β-RmAbs in killing tumor cells in cytolytic assays (Example 8; Table 1).For example, the BDA8 mAb in Group I does not act in combination withthe AGH1 mAb in Group I to promote tumor cell cytotoxicity. Likewise,the Group III BKA11 and CDH10 mAbs do not cooperate in a tumor cellcytotoxicity assay.

[0101]FIG. 5A-C shows the effects of administering representativeanti-LT-β-R mAbs alone and in pairwise combination to tumor cells incytotoxicity assays in the presence of IFN-γ as a LT-β-R activatingagent. The Group IV anti-LT-β-R mAb CBE11 used alone has a slightcytotoxic effect which is enhanced in combination with the Group II mAbBHA10 (FIG. 5A). CBE11 elicits a similar effect with the Group III mAbCDH10 (FIG. 5B).

[0102] The cytotoxicity caused by administering a combination ofanti-LT-β-R mAbs in solution is not peculiar to the HT29 tumor cellline. FIG. 5C shows that the Group I AGH1 mAb and the Group III CDH10mAb act synergistically in killing two different tumor cell lines (HT29cells and WiDr cells) derived from human adenocarcinoma tumors.

[0103] Summary of Anti-LT-β-R mAbs Characteristics

[0104] All of the anti-LT-β-R mAbs of this invention, when cross-linkedby immobilization, act as LT-β-R activating agents in the presence of asecond LT-β-R activating agent such as IFN-γ. The ability of anti-LT-β-RmAbs to act as LT-β-R activating agents in the presence or absence ofLT-α1/β2 in solution often varies according to the state of the cells atthe time of the test. Table 2, infra, summarizes the properties of theanti-LT-β-R mAbs characterized by this invention.

[0105] The Group I mAbs BDA8 and AGH1 do not function as LT-β-Ractivating agents in solution with LT-α1/β2. The BDA8 mAb actuallyblocks the anti-tumor effect of LT-α1/β2 (FIG. 3B and Table 2). Incontrast, the Group II anti-LT-β-R mAbs BCG6 and BHA10 have mixedagonistic and antagonistic effects when administered with LT-α1 /β2. TheGroup III anti-LT-β-R mAbs BKA11 and CDH10 are unique in their abilityto act as LT-β-R activating agents which potentiate anti-tumor effectsin the presence of LT-α1 /β2 and a second LT-β-R activating agent suchas IFN-γ without exhibiting the antagonistic effects often seen with theGroup II mAbs BCG6 and BHA10.

[0106] It is important to keep in mind that the classification of theanti-LT-β-R mAbs based on their ability to cooperate in tumor cellcytolytic assays reflects that they interact with distinct epitopes ofthe LT-β-R. The mAbs comprising a single group do not, however,necessarily have the same binding affinities for their cognate epitopes.Thus the variable results seen when comparing the effects of differentmAbs belonging to the same or different groups may represent differencesin binding affinities. Accordingly, it is possible that a Group I or aGroup IV mAb with a higher binding affinity for the LT-β-R could beisolated which would function like the Group III mAbs as a LT-β-Ractivating agent in the presence of LT-α1 /β2.

[0107] The hybridoma cell lines or subclones thereof which produce theanti-LT-β-R mAbs described above were deposited on Jan. 12, 1995 withthe American Type Culture Collection (ATCC) (Rockville, Md.) accordingto the provisions of the Budapest Treaty, and were assigned the ATCCaccession numbers designated as follows: ATCC CELL LINE mAb NameAccession No. a) AG.H1.5.1 AGH1 HB 11796 b) BD.A8.AB9 BDA8 HB 11798 c)BC.GE.AF5 BCG6 HB 11794 d) BH.A10 BHA10 HB 11795 e) BK.A11.AC10 BKA11 HB11799 f) CB.E11.1 CBE11 HB 11793 g) CD.H10.1 CDH10 HB 11797

[0108] All restrictions on the availability to the public of the aboveATCC deposits will be irrevocably removed upon the granting of a patenton this application.

[0109] Anti-LT-β-R IgM Monoclonal Antibodies Function as LT-β-RActivating Agents

[0110] Anti-LT-β-R mAbs which comprise more than the usual two IgGantigen binding sites will also function in solution as cell surfaceLT-β-R cross-linking agents, and will accordingly fall within thedefinition of a LT-β-R activating agent according to this invention. Theantigen binding sites of an anti-LT-β-R mAb can be built into IgMmolecules—which have ten antigen binding sites—using standardrecombinant DNA and hybridoma techniques (Example 12).

[0111] Alternatively, one may collect and enrich for complete mouse (orother animal) IgM molecules isolated by hybridoma fusion techniquesafter a single immunization with antigen. One way to enrich for IgMmolecules would be to immunize CD40 signaling-deficient mice (Kawabe etal., Immunity, 1, pp. 167-78 (1994); Xu et al., Immunity, 1, pp. 423-31(1994)). These mice cannot effectively produce IgGs and therefore theirresponse to challenge by antigen is enriched for IgM isotypes.

[0112] Anti-LT-β-R IgM antibodies, by virtue of their increased valency,can effectively aggregate LT-β-R molecules within the plane of themembrane, thereby enhancing LT-β-R signaling as compared to their IgGcounterparts having two antigen binding sites. A dramatic example of theincreased efficiency of multivalent antibodies in receptor clustering isseen with antibodies to the Fas receptor, where the IgM form is verypotent and normal bivalent IgGs are not effective in solution (Yoniharaand Yonihara, J. Exp. Med., 169, pp. 1747-56 (1989); Alderson et al.,Int. Immunol., 6, pp. 1799-1806 (1994)).

[0113] Likewise, the apo-1 mAb to the Fas receptor is an IgG3 mAb. ThismAb is a potent cytotoxic agent which relies on Fc interactions uniqueto IgG3 subtypes to aggregate into larger polyvalent forms,. Removal ofthe Fc region creates a F(ab)₂ form that cannot associate into largeraggregates and which is inactive (Dhein et al., J. Immunol., 149, pp.3166-73 (1992)). Thus by analogy, it is predicted that IgM versions ofanti-LT-β-R mAbs will be potent anti-tumor agents.

[0114] Anti-LT-β-R mAbs Inhibit Tumor Growth in Mice

[0115] The ability of a LT-β-R activating agent such as an anti-LT-β-RmAb to inhibit human tumor cell growth in vitro (Examples 6-8 and 13)may be indicative of in vivo anti-tumorigenic activity. Experimentsperformed in immunodeficient (SCID) mice demonstrate that an anti-LT-β-RmAb (CBE11) can efficiently block tumor formation by humanadenocarcinoma WiDr cells (Example 14; FIG. 6). Mice inoculatedsubcutaneously (s.c.) with WiDr cells form measurable tumors within twoweeks. When mice were treated i.p. with the CBE11 mAb at the same timeas the WiDr cells were inoculated s.c., tumor outgrowth was dramaticallyblocked (FIG. 6A). The anti-tumor action of the CBE11 anti-LT-β-R mAbwas enhanced by adding IFN-γ; CBE11 was effective, however, even withoutexogenous IFN-γ. In the CBE11+IFN-γ group, 7 of 16 animals completelylacked tumors, whereas the remaining animals had small nodules that hadnot progressed at 2 months. The CBE11 alone treated mice were similar tothe CBE11+IFN-γ group at 30 days. The CBE11 alone treated mice, however,eventually developed slowly growing tumors. There were statisticallysignificant differences between the CBE11 (+/−IFN-γ) groups and thecontrol groups (saline, IFN-γ alone and control anti-human LFA-3 mAb(1E6)+IFN-γ), but no significant differences among the control groups.The 1E6 and CBE11 mAbs are both IgG1 antibodies. The 1E6 mAb effectivelycoats the tumor cells but does not block tumor growth. Thus complementor natural killer cell-mediated events are not the sole basis for theanti-tumor activity of the CBE11 anti-LT-β-R mAb.

[0116] The efficacy of the CBE11 mAb in inhibiting tumor growth in vivoin the absence of exogenous IFN-γ was unexpected since there was adependence on IFN-γ for measurable LT-β-R-based in vitro cytotoxiceffects. Either there is some crossover of mouse IFN-γ onto human IFN-γreceptors, or other mechanisms may be involved in vivo.

[0117] The CBE11 anti-LT-β-R mAb can also inhibit growth of anestablished tumor in mice (FIG. 6B). Mice were inoculated s.c. with WiDrhuman adenocarcinoma cells at day 1 and tumors were allowed to developfor 15 days (Example 14). Tumors in animals treated i.p. with IFN-γalone, or with the control anti-human LFA-3 mAb (1E6)+IFN-γ, continuedto increase in size over the course of the 7-week experiment. Incontrast, tumors treated with the CBE11 anti-LT-β-R mAb (+IFN-γ oralone) stopped growing, and following three injections of CBE11 antibodyover a three week period, tumor growth was arrested out to 49 dayspost-inoculum when the experiment was terminated (FIG. 6B).

[0118] These experiments demonstrate that an anti-LT-β-R mAb whichactivates LT-β-R signaling can effectively inhibit tumor formation atearly stages and can also block continued tumor cell growth at laterstages of tumorigenesis in vivo. These experiments also demonstrate thatadministration of a single LT-β-R activating agent may be effective fortreating or reducing the advancement, severity or effects of neoplasiain an affected animal.

[0119] The procedures described in Example 14 may be used to identifyLT-β-R activating agents according to this invention which functionalone or in combination to inhibit tumor cell growth in vivo. It isenvisioned that other LT-β-R activating agents—including but not limitedto those identified using in vitro tumor cell cytotoxicity assays—mayhave similar anti-tumor effects in vivo when administered either aloneor in combination to animals or humans.

[0120] The Use of IFN-γ and Other LT-β-R Activating Agents

[0121] The cytotoxic effects of LT-α/β heteromeric complexes and ofcross-linked or multiple anti-LT-β-R Abs on tumor cells is enhanced bythe presence of a LT-β-R activating agent, particularly IFN-γ. Humanadenocarcinoma cells of intestinal origin (HT29 cells) have previouslybeen shown to be sensitive to FasR signaling (Yonehara and Yonehara, J.Exp. Med., 169, pp. 1747-56 (1989)); and to TNF and LT-α in the presenceof IFN-γ (Browning et al., J. Immunol., 143, pp. 1859-67 (1989)).

[0122] The amount of LT-β-R activating agent required to enhance theanti-tumor activity of LT-α/β heteromeric complexes, anti-LT-β-R Abs orother LT-β-R activating agents of this invention will depend on the cellor tissue type being treated, and also with the mode of treatment, andcan be determined empirically using routine procedures. The LT-β-Ractivating agent can be provided at a concentration or delivered at arate determined to be effective in conjunction with other LT-β-Ractivating agents administered, taking into consideration the factorslisted above.

[0123] Alternatively, endogenous LT-β-R activating agents such asinterferons like IFN-γ, which may be produced by cells or tissuesurrounding the target tumor cells, can be relied upon. Endogenous IFN-γis normally produced upon viral infection, and is also found in thevicinity of tumors (Dinge et al., Immunity, 1, pp. 447-56 (1994)).

[0124] Any agent which is capable of inducing interferons, preferablyIFN-γ, and which potentiates the cytotoxic effects of LT-α/β heteromericcomplexes and anti-LT-β-R mAbs on tumor cells falls within the group ofLT-β-R activating agents of this invention. While virus infectionnormally induces IFN-γ production, the levels of endogenous IFN-γ may beenhanced by other agents (Example 10). For example, clinical experimentshave demonstrated interferon induction by double stranded RNA (dsRNA)treatment. Accordingly, polyriboguanylic/polyribocytidylic acid(poly-rG/rC) and other forms of dsRNA are effective as interferoninducers (Juraskova et al., Eur. J. Pharmacol., 221, pp. 107-11 (1992)).

[0125] The interferon stimulator from Glycyrrhiza glabra (Acharya etal., Indian J. Med. Res., 98, pp. 69-74 (1993)), and pharmaceuticalagents, many of which are orally administrable, may also be used toboost endogenous interferon levels. Such interferon inducers include:imiquimod (Bernstein et al., Antiviral Res., 20, pp. 45-55 (1994));saparal (Paramonova et al., Vopr. Virusol., 39, pp. 131-34 (1994)); arylpyrimidones such as bropirimine (Onishi and Machida, Hinvokika Kiyo, 40,pp. 195-200 (1994)); Ridostin (Cheknev et al., Vopr. Virusol., 39, pp.125-28 (1994)).

[0126] Several of these interferon inducing agents have beencharacterized as inducers of type I interferons such as IFN-α. Type Iinterferons can also function as LT-β-R activating agents but are lesspotent than IFN-γ.

[0127] Treatments Using LT-α/β Complexes and LT-β-R Activating Agents

[0128] The compositions of this invention will be administered at aneffective dose to treat the particular clinical condition addressed.Determination of a preferred pharmaceutical formulation and atherapeutically efficient dose regiment for a given application is wellwithin the skill of the art taking into consideration, for example, thecondition and weight of the patient, the extent of desired treatment andthe tolerance of the patient for the treatment.

[0129] Typically, humans can tolerate up to 100-200 μg/m² of TNF beforeserious toxicity is manifested (Schiller et al., Cancer Res., 51, pp.1651-58 (1991)). In mice, dosages in the range of 1-5 μg/mouse/day givenwith 5×10⁴ units of recombinant human IFN-γ caused human primary tumorregression (Balkwill et al., CIBA Foundation Symposium (1987); Havell etal., J. Exp. Med., 167, pp. 1067-85 (1988)). Based on the relativeeffectiveness of TNF and LT-α1/β2 in the HT29 cytolytic assays,approximately 5-25 μg/mouse/day of LT-α1/β2 will provide a therapeuticdose range. Extrapolating to the human, it is expected that LT-α1/β2dosages of at least 1 mg/m² will be required in combination with anLT-β-R activating agent such as IFN-γ.

[0130] Historically, IFN-γ therapy has been undertaken either at maximumtolerated doses in the range of 100-250 μg/m², or at “immunomodulatory”levels in the range of 10-25 μg/m² (see e.g. Kopp et al., J.Immunother., 13, pp. 181-90 (1993)). Combination therapies with twointerferons have used 4×10⁶ units/m² of IFN-α and approximately 250μg/m² of IFN-γ (Niederle et al., Leuk. Lymohoma, 9, pp. 111-19 (1993)).Intermediate doses of about 25-100 μg/m² of IFN-γ in combination withthe LT-α/β heteromeric complexes or purified anti-LT-β-R-Abs describedherein are expected to be suitable starting points for optimizingtreatment doses.

[0131] Administration of the LT-α/β heteromeric complexes andcross-linked anti-LT-β-R Abs of this invention, including isolated andpurified forms of the antibodies or complexes, their salts orpharmaceutically acceptable derivatives thereof, may be accomplishedusing any of the conventionally accepted modes of administration ofagents which exhibit anti-tumor activity.

[0132] The pharmaceutical compositions used in these therapies may alsobe in a variety of forms. These include, for example, solid, semi-solidand liquid dosage forms such as tablets, pills, powders, liquidsolutions or suspensions, suppositories, and injectable and infusiblesolutions. The preferred form depends on the intended mode ofadministration and therapeutic application. Modes of administration mayinclude oral, parenteral, subcutaneous, intravenous, intralesional ortopical administration.

[0133] The LT-α/β heteromeric complexes, IFN-γ, and anti-LT-β-R Abs may,for example, be placed into sterile, isotonic formulations with orwithout cofactors which stimulate uptake or stability. The formulationis preferably liquid, or may be lyophilized powder. For example, the LTcomplexes and/or anti-LT-β-R Abs and IFN-γ may be diluted with aformulation buffer comprising 5.0 mg/ml citric acid monohydrate, 2.7mg/ml trisodium citrate, 41 mg/ml mannitol, 1 mg/ml glycine and 1 mg/mlpolysorbate 20. This solution can be lyophilized, stored underrefrigeration and reconstituted prior to administration with sterileWater-For-Injection (USP).

[0134] The compositions also will preferably include conventionalpharmaceutically acceptable carriers well known in the art (see forexample Remington's Pharmaceutical Sciences, 16th Edition, 1980, MacPublishing Company). Such pharmaceutically acceptable carriers mayinclude other medicinal agents, carriers, genetic carriers, adjuvants,excipients, etc., such as human serum albumin or plasma preparations.The compositions are preferably in the form of a unit dose and willusually be administered one or more times a day.

[0135] The pharmaceutical compositions of this invention may also beadministered using microspheres, liposomes, other microparticulatedelivery systems or sustained release formulations placed in, near, orotherwise in communication with affected tissues or the bloodstream.Suitable examples of sustained release carriers include semipermeablepolymer matrices in the form of shaped articles such as suppositories ormicrocapsules. Implantable or microcapsular sustained release matricesinclude polylactides (U.S. Pat. No. 3,773,319; EP 58,481), copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., iBiopolymers, 22, pp. 547-56 (1985)); poly(2-hydroxyethyl-methacrylate)or ethylene vinyl acetate (Langer et al., J. Biomed. Mater. Res., 15,pp. 167-277 (1981); Langer, Chem. Tech., 12, pp. 98-105 (1982)).

[0136] Liposomes containing LT-α/β heteromeric complexes and/oranti-LT-β-R Abs and IFN-γ can be prepared by well-known methods (See,e.g. DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. U.S.A., 82,pp. 3688-92 (1985); Hwang et al., Proc. Natl. Acad. Sci. U.S.A., 77, pp.4030-34 (1980); U.S. Pat. Nos. 4,485,045 and 4,544,545). Ordinarily theliposomes are of the small (about 200-800 Angstroms) unilamellar type inwhich the lipid content is greater than about 30 mol.% cholesterol. Theproportion of cholesterol is selected to control the optimal rate of LTcomplex and/or anti-LT-β-R Abs and IFN-γ release.

[0137] The LT-α/β heteromeric complexes and anti-LT-β-R Abs of thisinvention may also be attached to liposomes containing other LT-β-Ractivating agents, chemotherapeutic agents or IFN-γ to supplement theIFN-γ typically found in the region of tumors. Attachment of LTcomplexes and anti-LT-β-R Abs to liposomes may be accomplished by anyknown cross-linking agent such as heterobifunctional cross-linkingagents that have been widely used to couple toxins or chemotherapeuticagents to antibodies for targeted delivery. Conjugation to liposomes canalso be accomplished using the carbohydrate-directed cross-linkingreagent 4-(4-maleimidophenyl) butyric acid hydrazide (MPBH) (Duzgunes etal., J. Cell. Biochem. Abst. Suppl. 16E 77 (1992)).

[0138] Advantages of Therapeutic Compositions Based on Anti-LT-β-RActivation

[0139] An anti-tumor therapy based upon LT-β-R activation would haveseveral advantages. LT-β-R binds to LT-α/β heteromeric complexes withhigh affinity in β/β clefts, and with lower affinity in α/β cleftscreated at the interfaces between adjacent LT-α and LT-β subunits. Incontrast, the TNF receptors bind to LT-α/β heteromeric complexes withhigh affinity only in a/a clefts. Accordingly, purified LT-α1/β2complexes bind with high affinity to LT-β-R between adjacent LT-βsubunits, but lack α/α clefts and thus do not cross-activate signalingthrough the TNF receptors. Thus the LT-α/β heteromeric complexes of thisinvention will not stimulate TNF-associated inflammatory responses.

[0140] LT-α1/β2 administration does not activate endothelial cellchanges associated with inflammatory response even at relatively highlevels. For this reason, the side effects due to activation of theinflammatory cascades observed with TNF should not be a problem usingthe pharmaceutical compositions and treatment methods to activate theLT-β-R.

[0141] Human LT-α1/β2 binds to mouse LT-β-R essentially as well as tohuman LT-β-R. Injection of 100 μg human LT-α1/β2 per mouse is not lethal(Example 11), suggesting that stimulation of LT-β-R in the whole animaldoes not have the overt toxicity seen when similar experiments weretried with FasR or p60 TNF-R activation.

[0142] The use of specific anti-LT-β-R monoclonal antibodies or antibodycombinations to trigger this pathway in humans may have severaladvantages over treatment with LT-α/β heteromeric complexes. Ananti-receptor antibody therapy will be more selective than treating withligand. Moreover, recombinant forms of anti-LT-β-R mAbs would be easierto engineer and produce in large scale than the soluble LT-α/βheteromeric complexes.

[0143] It is envisioned that the mAbs or LT-α/β heteromeric complexeswould be administered to tumor-bearing people in conjunction with aconventional anti-tumor therapy (i.e. radiation and chemotherapy). Acombined treatment of LT-β-R activation with conventional chemotherapiesmay provide an extra factor of tumor killing activity that would be morelikely to clear a patient of tumorigenic cells than when conventionalanti-tumor therapy is used alone.

[0144] It is further possible that this approach may have relatively fewside effects and therefore could be given in a semi-prophylactic sensein cases of carcinomas that may not have metastasized, or in patientsfrom families who show a genetic pre-disposition for a certain type ofcancer.

[0145] The following are examples which illustrate the LT-α/βheteromeric complexes and the anti-LT-β-R mAbs of this invention and themethods used to characterize them. These examples should not beconstrued as limiting: the examples are included for purposes ofillustration and the present invention is limited only by the claims.

EXAMPLE 1 Generation of Baculovirus-infected Insect Cell SupernatantsContaining IT-α/β Forms

[0146] Recombinant baculovirus encoding either full length LT-α or asecreted form of LT-β were made as described (Crowe et al., Science,264, pp. 707-710 (1994)). High five insect cells (Invitrogen, San Diego,Calif.) were inoculated at a density of 2×10⁵ cells/ml into 7.2 litersof SF 900-II (Gibco) media without serum. The culture reached 1.8×10⁶cells/ml 48 hours later and was infected with 150 ml (3×10⁸ PFU/ml) ofLT-β and 300 ml of LT-α baculovirus stocks. Two days later, the culturewas harvested and the cell debris was removed by centrifugation. Afteraddition of EDTA and PMSF (1 mM EDTA and 150 μM PMSF finalconcentration), the clarified supernatant was concentrated 10-fold byultrafiltration using a S1YM10 (Amicon) spiral cartridge. Theconcentrate was divided into six 120 ml portions and aliquots werestored at −70 C prior to purification.

EXAMPLE 2 Preparation of Soluble LT-β Receptors as Immunoglobulin FcChimera

[0147] The extracellular domain of LT-β-R up to the transmembrane regionwas amplified by PCR from a cDNA clone using primers that incorporatedNotI and SalI restriction enzyme sites on the 5′ and 3′ ends,respectively (Browning et al., J. Immunol., 154, pp. 33-46 (1995)). Theamplified product was cut with NotI and SalI, purified and ligated intoNotI-linearized vector pMDR901 along with a SalI-NotI fragment encodingthe Fc region of human IgG1. The resultant vector contained thedihydrofolate reductase gene and the LT-β-R-Fc chimera driven byseparate promoters. The vector was electroporated into CHO dhfr⁻ cellsand methotrexate-resistant clones were isolated as per standardprocedures. The LT-β-R-Fc is secreted into the medium and an ELISA assaywas used to select for cell lines producing the highest level of thechimeric protein. A high-producing cell line was grown to large numbersand conditioned medium collected. The pure protein was isolated byProtein A sepharose fast flow affinity chromatography.

EXAMPLE 3 Affinity Purification of LT-α1/β2 Using TNF-R and LT-β-R

[0148] To prepare resins for the receptor affinity purification of LTforms, purified preparations of LT-β-R-Fc (as described in Example 2herein) and TNF-R p60-Fc (Crowe et al., Science, 264, pp. 707-10 (1994))were immobilized on CNBr-sepharose (Pharmacia) at 5 mg/ml resinessentially following the manufacturer's specifications. The resins wereput through one elution cycle prior to use. A portion (120 ml) of theS1Y10 concentrate was passed over two sequential p60 TNF-R-Fc columns,which bind LT-α and LT-α2/β1. The flow through, which contained LT-α1/β2and LT-β , was passed over a LT-β-R-Fc column. The column was washedwith 5 volumes each of PBS, PBS with 0.5 M NaCl and PBS, and then theLT-α and LT-α2/β1 complexes were eluted with 25 mM sodium phosphate, 100mM NaCl, pH 3.5. Elution fractions were immediately neutralized with{fraction (1/20)} volume of 0.5 M sodium phosphate, pH 8.6 and stored onice. Fractions containing protein were identified by absorbance at 280nm, peak fractions were pooled and the elution pools from the columnswere analyzed by SDS-PAGE stained with coomassie brilliant blue. Elutionas described above yielded greater than 95% pure LT-α1/β2.

EXAMPLE 4 Characterization of the Purified LT-α1/β2 Ligands

[0149] The fractions of Example 3 were sized by gel exclusionchromatography to assess whether trimers were formed and if aggregateswere present. A TSK G3000 sw ×2 column was used at a flow rate of 0.5ml/min to size separate a BioRad gel filtration protein standard, andthe three different LT trimers, LT-α3, LT-α2/β1 and LTα1/β2. FIG. 1Ashows that very little, if any of the LT-α1/β2 trimers show up as highmolecular weight aggregate. Comparison to size standards shows that thethree forms are all trimeric, i.e. about 50-60 kDa. Assuming the trimer,the stoichiometry of LT-α to LT-β contained in the purified LT-α1/β2 andLT-α2/β1 fractions was evaluated by either densitometry of the coomassiestained gels or by peak height analysis of the two peaks followingresolution on C4 reverse phase HPLC. Both measurements confirmed theidentity of the fractions eluted off the affinity columns as describedabove.

[0150] The purity of the preparations was further assessed by ionexchange chromatography using BioCAD instrumentation to run pH maps ofLT-α1/β2 and LT-α2/β1 on the weak cation exchanging resin under severaldifferent buffer systems. The method that exhibited the greatest abilityto cleanly retain and separate the three trimers incorporated a POROS CM(carboxymethyl) column run at 5 ml/min with a 16.66 mM MES, 16.66 mMHEPES, 16.66 mM Na Acetate pH 6.5 buffer and eluting with a 1 M NaClgradient over 20 column volumes. The BioCAD chromatograms of LT-α1/β2and LT-α2/β1 complexes are shown in FIG. 1B. Each trimer, LT-α3,LT-α2/β1 and LT-α1/β2 eluted at a different salt concentration and therewas no evidence for cross contamination of more than 1-2% in the variouspreparations.

EXAMPLE 5 Killing of HT29 Human Adenocarcinoma Cells by Soluble LT-α1/β2Complexes

[0151] The HT29 cytolytic assay has been previously described (Browningand Ribolini, J. Immunol., 143, pp. 1859-67 (1989)). In a typical assay,serial dilutions of LT-α1/β2 (and other cytokines where applicable) wereprepared in 0.05 ml in 96 well plates and 5000 trypsinized HT29-14 cellsadded in 0.05 ml of media containing 0 or 80 U/ml (anti-viral units) ofhuman IFN-γ. HT29-14 cells are from a subclone of the originalATCC-derived HT29 line that is more homogeneous. HT29-14 cells were usedin the assays; all of these results can also be observed using theoriginal ATCC-derived HT29 line. After 3-4 days, mitochondrial reductionof the dye MTT was measured as follows: 10 1 of MTT was added and after3 hours, the reduced dye dissolved with 0.09 ml of isopropanol with 10mM HCl, and the O.D. measured at 550 nm. Soluble receptor forms preparedas described herein, or pure human IgG were added in 10 μl prior to thecells to give a final concentration of 5 μg/ml.

[0152]FIG. 2A shows the killing of HT29 cells by treating with anti-Fasreceptor mAb CH-11 (which stimulates FasR signaling); TNF, LT-α3,LT-α1/β2 and LT-α2/β1 ligands in conjunction with IFN-γ. Visualinspection of the cells treated with LT-α1/β2 reveals that this agentkills cells rather than just blocking cell proliferation. In the absenceof IFN-γ, no effects are observed, reflecting the unusual ability ofIFN-γ to influence how cells interpret signaling from the TNF family ofreceptors. Interferons α and β were 100-fold less effective than IFN-γas quantitated based on anti-viral activity units.

[0153]FIG. 2B shows the inhibition of LT-α1/β2 killing by solubleLT-β-R-Fc but not p60-TNF-R-Fc, demonstrating that cytotoxicity isspecific to LT-α1/β2. The lack of inhibition by p60-TNF-R-Fc indicatesthat contaminating LT-α (known to be less than 1%) cannot account forthe cytotoxic activity of LT-α1/β2.

EXAMPLE 6 Anti-LT-β-R mAbs Potentiate the Killing of HT29 Cells byLT-α1/β2 Complexes

[0154] Cytolytic assays were performed as described in Example 5, exceptthat IFN-γ and anti-LT-β-R mAbs (0.01 -1000 ng/ml series) were added tothe cells at 2× final concentration and then 50 μl of the cell solutionwere added to the wells containing diluted LT-α1/β2. Growth was assessedas described in Example 5. FIG. 3 shows the differential effects of twodifferent anti-LT-β-R mAbs in their ability to potentiate LT-α1/β2cytotoxic activity. FIG. 3A shows that the anti-LT-β-R mAb CDH10potentiates LT-α1/β2 cytotoxic activity in a dosage-dependent manner.FIG. 3B shows the effects of another anti-LT-β-R mAb, BDA8, in the sameassay. The BDA8 mAb inhibits the cytotoxic activity of LT-α1/β2 ratherthan potentiating tumor cell death.

EXAMPLE 7 Immobilized Anti-LT-β-R mAbs can Kill HT29 Tumor Cells

[0155] To immobilize anti-LT-β-R mAbs onto a plastic surface, 96-welltissue culture plates were coated with 50 μl of 10 μg/ml goat anti-mouseFc polyclonal antibody (Jackson ImmunoResearch), washed and blocked with5% FCS in PBS, followed by capture of the indicated anti-LT-β-R mAb andanother wash. HT29 cells were plated into the mAb-coated wells, andcytolytic assays were conducted as in Example 5. FIG. 4A illustrates thecytotoxic effects of immobilized BDA8 and CDH10 anti-LT-β-R mAbs on HT29cells. Each mAb individually elicits cytotoxicity on tumor cells when itis immobilized onto a surface. FIG. 4B shows that the same BDA8 andCDH10 anti-LT-β-R mAbs tested individually in solution are not cytotoxicand thus the cytolytic activity of a single anti-LT-β-R mAb in vitroappears to be a function of its immobilization.

EXAMPLE 8 A Combination of Anti-LT-β-R mAbs in Solution Directed AgainstDistinct Epitopes Kill HT29 Cells

[0156] Growth of HT29 cells was assessed as described in Example 5except that either one or two anti-LT-β-R mAbs were included in thegrowth medium. Table 1 shows the effects on HT29 cells observed whenvarious anti-LT-β-R mAbs were included in solution (i.e., notimmobilized on plastic). The anti-LT-β-R mAbs can be arranged intogroups I-IV based on their relative abilities to work in combinationwith each other in a HT29 cytolytic assay. The anti-LT-β-R mAbs resultsgenerated in cytolytic assays parallel receptor binding data whichsuggest that the mAbs in each different group recognize differentepitopes of the LT-β-R. Table 1. Combinations of soluble anti-LT-β-RmAbs are cytotoxic to human adenocarcinoma HT29 cells. Anti-LT-β-R mAbsare grouped into Groups I, II, III and IV based on their effects incombination with each other in HT29 cell cytolytic assays. Pluses referto the relative level of cytolytic effects of the mAb combination onHT29 cells in the presence of 80 U/ml IFN-γ. nr=not relevant; nd=notdetermined. Second mAb First Group I Group II Group III Group IV GroupmAb BDA8 AGH1 BCG6 BHA10 BKA11 CDH10 CBE11 I BDA8 nr − + ++ + nd nd AGH1− nr ++ +++ ++ nd nd II BCG6 ++ ++ nr − +++ nd nd BHA10 ++ +++ − nr ++++ ++++ III BKA11 + ++ +++ nd nr − nd CDH10 ++ ++ ++ +++ − nr +++ IVCBE11 nd + + ++++ nd ++++ nr

[0157] FIGS. 5A-D quantitates the effects of including in the HT29cytolytic assay representative pairwise combinations of cooperatinganti-LT-β-R mAbs directed against different epitopes of LT-β-R. FIG. 5Ashows the cytotoxic effects of BHA10 and CBE11 , FIG. 5B of CDH10 andCBE11 , and FIG. 5C of CDH10 and AGH1, alone and in combination. FIG. 5Dshows the cytotoxic effects of the CDH10 and AGH1 mAb combination in adifferent tumor cell line called WiDr.

[0158] Table 2 summarizes the characteristics of the representativeanti-LT-β-R mAbs of this invention. TABLE 2 Summary of Mouse Anti-humanLT-β-R mAbs HT29 Cytotoxicity Blocking mAb Soluble Soluble mAb CellReceptor Immobilized mAb mAb with Group mAb Name Staining^(a)Binding^(b) on Plastic^(c) alone Ltα1β1 I BDA8 +++ +++ + +/−^(d)inhibits I AGH1 +++ +++ +/− inhibits II BCG6 +++ ++ + +/− mixed II BHA10+++ +++ + +/− mixed III BKA11 +++ +/− + − potentiates III CDH10 ++++/− + +/− potentiates IV CBE11 +++ +++ + +/− no effect Controls MOPC21 −− − − no effect HT29/26 − nd − − no effect TS 2/9^(e) nd nd − − noeffect

EXAMPLE 9 Reliance on Endogenous IFN-γ for the Treatment of Tumor Cells

[0159] IFN-γ, a preferred LT-β-R activating agent of the presentinvention, is a cytokine which exhibits anti-tumor activity and which istolerated in humans. Endogenous IFN-γ present in the environmentsurrounding a tumor may be at sufficiently high concentrations tofunction as a LT-β-R activating agent of this invention without addingexogenous IFN-γ. The concentration of IFN-γ in the vicinity of a tumormay be ascertained using standard immunochemical techniques with tissuesamples from the region of the tumor. If the endogenous concentration ofIFN-γ is high enough to elicit anti-tumor activity in combination withthe LT-α/β heteromeric complexes or anti-LT-β-R mAbs of this invention(as determined by the cytolytic assays described herein), then IFN-γneed not be administered as a second LT-β-R activating agent in thecompositions or methods of this invention.

EXAMPLE 10 Induction of Endogenous IFN-γ as a LT-β-R Activating Agentfor the Treatment of Tumor Cells

[0160] Compounds which can induce the endogenous production ofinterferons such as IFN-γ fall within the group of LT-β-R activatingagents of this invention. For example, interferons can be induced bytreating with double-stranded RNA molecules such aspolyribo-guanylic/polyribocytidylic acid (poly-G/C).

[0161] Female C57/b16 (6-8 weeks old) can be injected with 18 mg (600mg/kg) of D-galactosamine which sensitizes mice to the effects of TNFand other anti-tumor agents. A series of concentrations of poly-G/C(Juraskova et al., Eur. J. Pharmacol., 221, pp. 107-11 (1992)) in aneutral saline solution is added to purified LT-α1/β2 (10-100μg) and thesolution administered to mice as an intraperitoneal (i.p.) injection.The anti-tumor activity of LT-α1/β2 will be enhanced by the presence ofpoly-rG/rC double stranded RNA.

[0162] Similarly, the interferon stimulator from the plant Glycyrrhizaglabra (Acharya et al., Indian J. Med. Res., 98, pp. 69-74 (1993)) maybe administered to humans intravenously at doses ranging from 40-100ml/day. The optimal dose for LT-β-R activation in the presence of eitherLT-α/β heteromeric complexes or anti-LT-β-R Abs may be determinedempirically and will depend on factors such as the tumor type, mode ofdelivery and delivery schedule.

[0163] Imiquimod R-837 (Bernstein et al., Antiviral Res., 20, pp. 45-55(1994)); Saparal (Paramonova et al., i Vopr. Virusol., 39, pp. 131-34(1994)); Bropirimine (Onishi and Machida, Hinvokika Kiyo, 40, pp.195-200 (1994)); or Ridostin (Cheknev et al., Vopr. Virusol., 39, pp.125-28 (1994)), may also be administered as LT-β-R activating agents inconjunction with LT-α/β heteromeric complexes, anti-LT-β-R Abs, or acombination thereof. In each case, the preferred modes of delivery andoptimal doses can be determined empirically using the published reportsas starting points for optimization by routine clinical procedures.

EXAMPLE 11 Mice Tolerate Injections of Human LT-α1/β2

[0164] Female C57/b16 (6-8 weeks old) acclimated to the facility forseveral days were injected i.p. with 18 mg (600 mg/kg) ofD-galactosamine, which sensitizes mice to the effects of TNF and otheranti-tumor agents. Either human TNF, LT-α or LT-α1/β2 was then giveni.p. Table 3 documents the survival of treated mice 24 hours afterinjection. TABLE 3 Agent Dose (μg/animal) Survival saline — 4/4 hu-TNF0.2 0/6 hu-TNF 1.0 0/2 hu-TNF 10 0/4 hu-LT-α 0.2 2/2 hu-LT-α 1.0 2/2hu-LT-α1/β2 10 2/2 hu-LT-α1/β2 100 2/2

EXAMPLE 12 Construction of a Recombinant Anti-LT-β-R IgM MonoclonalAntibody

[0165] Using the anti-tumor cytotoxicity assays described above coupledwith standard tumor growth models in immunodeficient mice, ananti-LT-β-R IgG with suitable properties can be selected. Universalprimers which hybridize to each of the variable domains of the IgG heavyand light chains of the selected anti-LT-β-R IgG can be used to preparevariable domain DNA from RNA isolated from the secreting hybridoma cellline using standard reverse transcriptase/PCR methodologies. Theseprotocols have been described (Arulanandam et al., J. Exp. Med., 177,pp. 1439-50 (1993); Lane et al., Eur. J. Immunol., 22, pp. 2573-78(1993); Traunecker et al., Nature, 339, pp. 68-70 (1989)).

[0166] The amplified products are then assembled into vectors containingthe human CH1, CH2 and CH3μ chain domains. Co-expression of the twochains in a single host will allow assembly of the heavy and lightchains into a pentameric IgM molecule. This molecule is a chimeracomposed of mouse variable regions coupled to human constant regions.

[0167] Alternatively, a process using PCR to amplify DNA encoding onlythe actual binding regions of the variable regions can be used.Amplified DNA is then inserted into vectors containing all of the humanIgG sequences except for the actual amino acids involved in binding theantigen. Such constructs are called “humanized” antibodies and thedetailed methods for their production are well-known (e.g. WO 94/04679).

EXAMPLE 13 Anti-LT-β-R IgM Monoclonal Antibodies Function as LT-β-RActivating Agents

[0168] Anti-LT-β-R IgM antibodies can be prepared in a recombinant formas described in Example 12. Alternatively, complete mouse IgMs isolatedby hybridoma fusion techniques using primary immunization of normal miceor extensive immunization of CD40 signaling-deficient mice (Kawabe etal., Immunity, 1, pp. 167-78 (1994); Xu et al., Immunity, 1, pp. 423-31(1994)) can be used as a source of anti-LT-β-R IgM mAbs.

[0169] Anti-LT-β-R IgM mAbs will be significantly more potent as LT-β-Ractivating agents than their normal bivalent IgG counterparts asmeasured by dose-response comparisons in the HT29 cytolytic assay in thepresence of IFN-γ. The anti-LT-β-R IgM mAbs function as LT-β-Ractivating agents both when they are immobilized and when they areadministered in solution. In addition, we expect that they will augmentthe anti-tumor activity of LT-α/β heteromeric complexes.

EXAMPLE 14 Anti-LT-β-R Monoclonal Antibodies Inhibit the Growth of HumanTumor Cells in SCID Mice

[0170] Balb/c SCID mice (Jackson Labs, Bar Harbor, Me.) were injectedwith 1×10⁶ trypsinized and washed human adenocarcinoma WiDr cells in avolume of 0.2 ml of PBS subcutaneously (s.c.) onto the back of theanimal. Injected WiDr cells form tumors in the mice, and the ability ofan anti-LT-β-R mAb to inhibit tumor growth was monitored. In one set ofexperiments, mice were treated with or without the CBE11 anti-LT-β-RmAb—either with or without human IFN-γ (106 antiviral units/mouse)—atthe same time as the WiDr cells were inoculated s.c. (FIG. 6A).Antibodies and IFN-γ were administered alone or together by i.p.injection in 0.2 ml. Control mice were injected with saline alone, IFN-γalone, or a control anti-human LFA-3 mAb (1E6) with IFN-γ. The size ofeach resulting tumor was scored 30 days after inoculation. Tumor volume(in cc) was calculated from the radius as determined by calipermeasurements in two dimensions. Animals treated with CBE11: or 1E6 mAbsreceived 10 μg/mouse or 50 μg/mouse of antibody (FIG. 6A; circles withdots and open circles, respectively).

[0171] In another set of experiments, mice were inoculated s.c. with theWiDr cells and tumors were allowed to grow for 15 days before the micewere treated with the CBE11 anti-LT-β-R mAb (FIG. 6B). At day 15 (beforeantibody treatment), the average tumor volume was 0.076 cc with anaverage diameter of 0.53 cm. The CBE11 anti-LT-β-R mAb (50 μg) was thenadministered—either with or without human IFN-γ (10⁶ antiviralunits/mouse)—by i.p. injection in 0.2 ml to a group of 12 animals.Injections were repeated three more times over a three week period.Control groups (12 mice/group) were injected with IFN-γ alone (10⁶antiviral units/mouse) or with 50 μg of a control anti-human LFA,-3 mAb(1E6)+IFN-γ (10⁶ antiviral units/mouse). The growth of the tumorspresent at day 15 was scored over time, from 15 to 49 days post-tumorcell inoculation. The results shown in FIG. 6B were determined in ablinded format. Tumors treated with CBE11 mAb either with or withoutIFN-γ stopped growing. Following three injections of CBE11 mAb(+/−IFN-γ) over three weeks, tumor growth was arrested for at least 7weeks post-inoculum, at which time the experiment was terminated.

What is claimed is:
 1. A method for treating or reducing theadvancement, severity or effects of neoplasia comprising the step ofadministering a therapeutically effective amount of a LT-α/β heteromericcomplex and a pharmaceutically acceptable carrier.
 2. The methodaccording to claim 1, wherein the LT-α/β heteromeric complex has aLT-α1/β2 stoichiometry.
 3. The method according to claim 1, wherein theLT-α/β heteromeric complex is a soluble LT-α/β heteromeric complex. 4.The method according to any one of claims 1-3, wherein the LT-α subunitis selected from the group consisting of lymphotoxin-α, native human oranimal lymphotoxin-α, recombinant lymphotoxin-α, soluble lymphotoxin-α,secreted lymphotoxin-α, lymphotoxin-α muteins, or lymphotoxin-α-activefragments of any of the above.
 5. The method according to any one ofclaims 1-3, wherein the LT-β subunit is selected from the groupconsisting of lymphotoxin-β, native human or animal lymphotoxin-β,recombinant lymphotoxin-β, soluble lymphotoxin-β, secretedlymphotoxin-β, lymphotoxin-β muteins, or lymphotoxin-β-active fragmentsof any of the above.
 6. The method according to claim 3, wherein theLT-β subunit is cleaved between amino acids 44 and 88 and the N-terminalportion replaced with a type I leader sequence.
 7. The method accordingto claim 6, wherein the type I leader sequence is the vascular celladhesion molecule 1 (VCAM-1) leader sequence.
 8. The method according toany one of claims 1-3, wherein the LT-α/β heteromeric complex isadministered in the presence of a therapeutically effective amount of atleast one LT-β-R activating agent.
 9. The method according to claim 8,wherein one LT-β-R activating agent comprises a therapeuticallyeffective amount of IFN-γ.
 10. The method according to claim 9, whereina second LT-β-R activating agent comprises a therapeutically effectiveamount of an anti-LT-β-R antibody.
 11. The method according to claim 10,wherein the anti-LT-β-R antibody is a monoclonal antibody.
 12. Themethod according to claim 11, wherein the anti-LT-β-R monoclonalantibody is selected from the group consisting of anti-LT-β-R mAb BKA11,CDH10, BCG6 and BHA10.
 13. A method for treating or reducing theadvancement, severity or effects of neoplasia comprising the step ofadministering a therapeutically effective amount of at least two LT-β-Ractivating agents and a pharmaceutically acceptable carrier.
 14. Themethod according to claim 13, wherein at least one LT-β-R activatingagent comprises an anti-LT-β-R antibody.
 15. The method according toclaim 14, wherein the anti-LT-β-R antibody is CBE11.
 16. The methodaccording to claim 13, wherein the LT-β-R activating agents comprise atleast two anti-LT-β-R monoclonal antibodies which are directed againstnon-overlapping epitopes of LT-β-R.
 17. The method according to claim16, wherein one anti-LT-β-R monoclonal antibody is selected from thegroup consisting of AGH1 and BDA8, and another anti-LT-β-R monoclonalantibody is selected from the group consisting of BCG6, BHA10, BKA11,CDH10 and CBE11.
 18. The method according to claim 16, wherein oneanti-LT-β-R monoclonal antibody is selected from the group consisting ofBCG6 and BHA10, and another anti-LT-β-R monoclonal antibody is selectedfrom the group consisting of AGH1, BDA8, BKA11, CDH10 and CBE11.
 19. Themethod according to claim 16, wherein one anti-LT-β-R monoclonalantibody is selected from the group consisting of BKA11 and CDH10, andanother anti-LT-β-R monoclonal antibody is selected from the groupconsisting of AGH1 and BDA8, BCG6, BHA10, and CBE11.
 20. The methodaccording to claim 16, wherein one anti-LT-β-R monoclonal antibody isCBE11, and another anti-LT-β-R monoclonal antibody is selected from thegroup consisting of AGH1, BDA8, BCG6, BHA10, BKA11, CDH10 and CBE11. 21.The method according to claim 16, wherein the anti-LT-β-R monoclonalantibodies are CBE11 and BHA10.
 22. The method according to claim 16,wherein the anti-LT-β-R monoclonal antibodies are CBE11 and CDH10. 23.The method according to claim 16, wherein the anti-LT-β-R monoclonalantibodies are AGH1 and CDH10.
 24. The method according to any one ofclaims 13-23, wherein one LT-β-R activating agent is IFN-γ.
 25. A methodfor treating or reducing the advancement, severity or effects ofneoplasia comprising the step of administering a therapeuticallyeffective amount of cross-linked anti-LT-β-R antibodies as a firstLT-β-R activating agent in the presence of a second LT-β-R activatingagent and a pharmaceutically acceptable carrier.
 26. The methodaccording to claim 25, wherein the cross-linked anti-LT-β-R antibodiesare non-covalently immobilized on a surface.
 27. The method according toclaim 25, wherein the cross-linked anti-LT-β-R antibodies are covalentlyimmobilized on a surface.
 28. The method according to claim 25, whereinthe cross-linked anti-LT-β-R antibodies are non-covalently aggregated insolution by means of an anti-LT-β-R antibody cross-linking agent. 29.The method according to claim 28, wherein the anti-LT-β-R antibodycross-linking agent comprises a secondary antibody directed against theanti-LT-β-R antibody.
 30. The method according to claim 28, wherein theanti-LT-β-R antibody cross-linking agent comprises an Fc receptor whichbinds to the anti-LT-β-R antibody.
 31. The method according to claim 25,wherein the cross-linked anti-LT-β-R antibodies are covalentlyaggregated in solution by means of a chemical anti-LT-β-R antibodycross-linking agent.
 32. The method according to any one of claims25-31, wherein the second LT-β-R activating agent comprises IFN-γ.
 33. Amethod for treating or reducing the advancement, severity or effects ofneoplasia comprising the step of administering a therapeuticallyeffective amount of at least one LT-β-R activating agent and apharmaceutically acceptable carrier.
 34. The method according to claim33, wherein at least one LT-β-R activating agent comprises ananti-LT-β-R antibody.
 35. The method according to claim 34, wherein theanti-LT-β-R antibody is CBE11.
 36. A method for selecting a LT-β-Ractivating agent which acts in the presence of LT-α/β heteromericcomplexes comprising the steps of: a) culturing tumor cells in thepresence of LT-α/β heteromeric complexes, an effective amount of a firstLT-β-R activating agent and a second putative LT-β-R activating agent;and b) determining whether the second putative LT-β-R activating agentincreases the anti-tumor activity of the LT-α/β heteromeric complex inthe presence of the first LT-β-R activating agent.
 37. The methodaccording to claim 36, wherein the first LT-β-R activating agent isIFN-γ.
 38. The method according to claim 36, wherein the LT-α/βheteromeric complex has a LT-α1/β2 stoichiometry.
 39. A pharmaceuticalcomposition comprising a therapeutically effective amount of a LT-α/βheteromeric complex and a pharmaceutically acceptable carrier.
 40. Thepharmaceutical composition according to claim 39, wherein the LT-α/βheteromeric complex has a LT-α1/β2 stoichiometry.
 41. The pharmaceuticalcomposition according to claim 39, wherein the LT-α/β heteromericcomplex is soluble.
 42. The pharmaceutical composition according to anyone of claims 39-41, further comprising a therapeutically effectiveamount of at least one LT-β-R activating agent.
 43. The pharmaceuticalcomposition according to claim 42, wherein one LT-β-R activating agentis IFN-γ.
 44. The pharmaceutical composition according to claim 42,wherein one LT-β-R activating agent is an anti-LT-β-R antibody.
 45. Thepharmaceutical composition according to claim 44, wherein theanti-LT-β-R antibody is a monoclonal antibody.
 46. The pharmaceuticalcomposition according to claim 45, wherein the anti-LT-β-R monoclonalantibody is selected from the group consisting of anti-LT-β-R mAb BKA11,CDH10, BCG6, and BHA10.
 47. A pharmaceutical composition comprising atherapeutically effective amount of at least two LT-β-R activatingagents without exogenous LT-α/β heteromeric complex, and apharmaceutically acceptable carrier.
 48. The pharmaceutical compositionaccording to claim 47, wherein at least one LT-β-R activating agentcomprises an anti-LT-β-R antibody.
 49. The pharmaceutical compositionaccording to claim 48, wherein the anti-LT-β-R antibody is a monoclonalantibody.
 50. The pharmaceutical composition according to claim 49,wherein the anti-LT-β-R monoclonal antibody is CBE11.
 51. Thepharmaceutical composition according to claim 47, wherein at least twoLT-β-R activating agents comprise anti-LT-β-R monoclonal antibodieswhich are directed against non-overlapping epitopes of LT-β-R.
 52. Thepharmaceutical composition according to claim 51, wherein oneanti-LT-β-R monoclonal antibody is selected from the group consisting ofAGH1 and BDA8, and another anti-LT-β-R monoclonal antibody is selectedfrom the group consisting of BCG6, BHA10, BKA11, CDH10 and CBE11. 53.The pharmaceutical composition according to claim 51, wherein oneanti-LT-β-R monoclonal antibody is selected from the group consisting ofBCG6 and BHA10, and another anti-LT-β-R monoclonal antibody is selectedfrom the group consisting of AGH1, BDA8, BKA11, CDH10 and CBE11.
 54. Thepharmaceutical composition according to claim 51, wherein oneanti-LT-β-R monoclonal antibody is selected from the group consisting ofBKA11 and CDH10, and another anti-LT-β-R monoclonal antibody is selectedfrom the group consisting of AGH1 and BDA8, BCG6, BHA10, and CBE11. 55.The pharmaceutical composition according to claim 51, wherein oneanti-LT-β-R monoclonal antibody is CBE11, and another anti-LT-β-Rmonoclonal antibody is selected from the group consisting of AGH1, BDA8,BCG6, BHA10, BKA11, CDH10 and CBE11.
 56. The pharmaceutical compositionaccording to claim 51, wherein the anti-LT-β-R monoclonal antibodies areCBE11 and BHA10.
 57. The pharmaceutical composition according to claim51, wherein the anti-LT-β-R monoclonal antibodies are CBE11 and CDH10.58. The pharmaceutical composition according to claim 51, wherein theanti-LT-β-R monoclonal antibodies are AGH1 and CDH10.
 59. Thepharmaceutical composition according to any one of claims 51-58, furthercomprising IFN-γ as one of the LT-β-R activating agents.
 60. Apharmaceutical composition comprising a therapeutically effective amountof cross-linked anti-LT-β-R antibodies as a LT-β-R activating agent anda pharmaceutically acceptable carrier.
 61. The pharmaceuticalcomposition according to claim 60, wherein the cross-linked anti-LT-β-Rantibodies are non-covalently immobilized on a surface.
 62. Thepharmaceutical composition according to claim 60, wherein thecross-linked anti-LT-β-R antibodies are covalently immobilized on asurface.
 63. The pharmaceutical composition according to claim 60,wherein the cross-linked anti-LT-β-R antibodies are non-covalentlyaggregated in solution by means of an anti-LT-β-R antibody cross-linkingagent.
 64. The pharmaceutical composition according to claim 63, whereinthe anti-LT-β-R antibody cross-linking agent comprises a secondaryantibody directed against the anti-LT-β-R antibody.
 65. Thepharmaceutical composition according to claim 60, wherein thecross-linked anti-LT-β-R antibodies are covalently aggregated insolution by means of a chemical anti-LT-β-R antibody cross-linkingagent.
 66. The pharmaceutical composition according to any one of claims60-65, further comprising IFN-γ as a second LT-β-R activating agent. 67.A pharmaceutical composition comprising a therapeutically effectiveamount of at least one LT-β-R activating agent without exogenous LT-α/βheteromeric complex, and a pharmaceutically acceptable carrier.
 68. Thepharmaceutical composition according to claim 67, wherein at least oneLT-β-R activating agent comprises an anti-LT-β-R antibody.
 69. Thepharmaceutical composition according to claim 68, wherein theanti-LT-β-R antibody is CBE11.
 70. An LT-β-R activating agent selectedaccording to the method of claim 36.